Method for the preparation of 1,5-bis(4-hydroxy-3-metoxy-phenyl)-penta-1,4-dien-3-one and derivatives with antitumoral properties

ABSTRACT

This patent of invention reports the method for the preparation of 1,5-bis(4-hydroxy-3-methoxyphenyl)-penta-1,4-dien-3-one and derivatives with antitumoral properties: the sample denominated 37 compound was obtained with high yield and purity with ultrasonic technique presenting cytostatic activity (growth inhibition) in the concentrations evaluated and cytotoxic activity (cellular death) from the concentration of 0.25 mg/mL against nine different types of human cancer cell lines. This compound has a LD 50 , equals to 8.54 g/Kg. That means this product can be considered itself as practically nontoxic. Doxorubicin, anticarcinogen medicine used as reference in all these tests, is a product extremely toxic (LD 50  of 20 mg/Kg) and it does not inhibit the growth of Mama NCI-ADR cell line (the one that expresses the phenotype of resistance against multiple drugs), therefore, out product presented a strong cytostatic activity. Other derivatives also presented a strong cytostatic activity, especially the one denominated EHB1 compound.

TECHNICAL FIELD

The present patent of invention refers to a NEW METHOD FOR THEPREPARATION OF 1,5-BIS(4-HYDROXY-3-METHOXYPHENYL)-PENTA-1,4-DIEN-3-ONEAND DERIVATIVES WITH ANTITUMORAL ACTIVITIES.

BACKGROUND

Bibliographical background about1,5-bis(4-hydroxy-3-methoxyphenyl)-penta-1,4-dien-3-one compound andderivatives and its method for the preparation.

The compound denominated1,5-bis(4-hydroxy-3-methoxyphenyl)-penta-1,4-dien-3-one is known sincethe year 1927 since E. Glaser and E. Tramer for the first time reportedits synthesis with a 60% yield (Journal für praktische Chemie, 116,331-346, 1927) from the vanillin and acetone in the presence ofconcentrated hydrochloric acid, used as a catalyzer.

Further, P. Ramanan and M. Rao synthesized this product in 1989 (IndianJournal Pharm. Sci., 51, 207, 1989) from 4-O-methoxymethylvanillin andacetone in an alkaline medium, obtaining a yield of 42% after purifyingthe same using the thin-layer chromatograph (silica gel).

In 1997, a group integrated by S. Sardjiman, et al. (Eur. Journal Med.Chem. 32, 625-630, 1997) developed a new variant synthesis usingequimolecular quantities of vanillin and acetone in the presence ofconcentrated hydrochloric acid, reporting a raw yield of 89% (withoutpurifying). For this reason, the melting point indicated in thisprocedure was 58° C. less than the one reported by Glaser and Tramer.

M. Artico et al. also obtained this substance one year later (JournalMed. Chem. 41, 3948-3960, 1998) only obtaining a poor yield of 18%. Themelting temperature (114-116° C.) is lower than the one reported byGlaser and Tramer, what is makes think that the compound was notobtained pure in despite of the use of the chromatograph of column.

Additionally, we can find in the bibliographic revision the article ofthe American patent (U.S. Pat. No. 4,521,629 of Jun. 4, 1985) of N.Cortese et al. entitled: “Method for the preparation of1,5-bis-aryl-1,4-pentadien-3-ones.” This invention reports to a methodof preparing certain bis-arylpentadienones containing fluorine, thatwere used as intermediate compounds for the preparation of insecticidalsubstituted amidinohydrazones, but it does not protect the products thatappear in our patent request.

Furthermore, the following documents of other patents related to thisfamily of organic compounds were found:

1-“Hair tonics containing bis(hydroxyphenyl)pentadienones.” Authors ofthe patent: Morita, Kazuyoshi; Hamada, Kazuto. Company: Kanebo, Ltd,Japan. Country: Jpn. Kokai Tokyo Koho, 7 pp. Idiom: Japanese. CA-Number:134:183278. PI: JP 2001048756, A2 20010220 JP 1999-224982 19990809.

2-“Skin-lightening cosmetics containing distyryl ketones.” Author:Morita, Kazuyoshi. Company: Kanebo, Ltd., Japan. Country: Jpn. KokaiTokyo Koho, 7 pp. Idiom: Japanese. CA-Number: 131:149078. PI: JP11209235 A2 19990803 JP 1998-10414 19980122.

3-“Acidic planting baths and methods for electrodepositing bright andductile zinc-nickel allows and additive composition for these baths.”Company: McGean-Rohco, Inc., USA. Author: Canaris, Valerie M. Country:U.S., 8 pp. Idiom: English. CA-Number: 111:183131. PI: US 4832802 A19890523 US 1988-206017 19880610 EP 346161 A1 19891213 EP 1989-30592519890612.

4-“Photopolymerizable compositions.” Compound: Eastman Kodak Co., USA.Authors: Noonan, John M.; McConkey, Robert C.; Arcesi, J. A.; Rauner:Frederick J. Country: Brit., 19 pp. Idiom: English PI: GB 1425476 A19760218 GB 1973-3986 19730322 US 3748133 A 19730724 US 1972-23792919720324.

None of these four patents, either directly or indirectly, are relatedto the antiproliferate properties shown by1,5-bits(4-hydroxy-3-methoxyphenyl)-penta-1,4-dien-3-one and itsderivatives.

Taking into account the bibliographic revision performed by the companyBypropind Patents and Trademarks based on the tracking in the ChemicalAbstracts collection, of Lifescience, of Biomed of Fiocruz library andby us in the Beilstein collection, we have concluded the there is nopatent of that compound and its derivatives applicable in the cancertreatment or even the ultrasonic technique in despite of the compoundhas been synthesized in the year of 1927 and so that the patent requestis possible and legitimate.

SUMMARY OF THE INVENTION

The present patent of invention reports the antitumoral properties of1,5-bis(4-hydroxy-3-methoxyphenyl)-penta-1,4-dien-3-one and derivativesand its procedure of preparing.

The sample denominated 37/01 compound was obtained with high yield andpurity by the ultrasonic technique presenting cytostatic activity(growth inhibition) in the concentrations evaluated and killed cellactivity (cellular death) from the concentration of 0.25 μg/mL againstnine different types of human cancer. This compound has a LD₅₀, equalsto 8.54 g/Kg. That means this product can be considered itself aspractically nontoxic. Doxorubicin, anticarcinogen medicine used asreference in all of these tests, is a product extremely toxic (LD₅₀ of20 mg/Kg) and its does not inhibit the growth of the Mama NCI-ADR celllines (the one that expresses the phenotype of resistance againstmultiple drugs), therefore our product presented a strong cytostaticactivity.

-   37 :R¹-CH₃: R²-H; R³-H; R⁴-H; R⁵═H; X-O-   EHB1:R¹═CH₃; R²═H₃CCO; R³═H; R⁴═H; R⁵═H; X═O-   HB6:R¹═CH₃; R²═CH₂CH═C(CH₃)₂; R³═H; R⁴═H; R⁵-H; X-O-   HBM1:R¹═CH₃; R²═CH₃; R³═H; R⁴═H; R⁵═H; X═O-   HB5:R¹═CH₃; R²═H; R³═H; R⁴═H; R⁵═H; X═C(CN)₂-   HB10:R¹═CH₃; R²═H; R³═CH₂CH═C(CH₃)₂; R⁴═H; R⁵═H; X═O-   HB11:R¹═CH₃; R²═H; R³═H; R⁴═H; R⁵═H; X═C(CN)CO₂C₂H₅-   HB12:R¹═H; R²═H; R³═H; R⁴═H; R⁵═H; X═O-   HB13:R¹═CH₃; R²═H; R³═Br; R⁴═H; R⁵═H; X═O-   HB14:R¹═CH₃: R²═CH₂CH═C(CH₃)₂; R³═Br; R⁴═H; R⁵-H; X═O-   HB15:R¹═H; R²═H; R³═CH₂CH═C(CH₃)₂; R⁴═CH₂CH═C(CH₃)₂; R⁵═H; X═O

Antitumoral activity shown by synthesized compounds is shown in FIG. 1.

The following designations are utilized in FIG. 1: Legend: NCI460 (Lungtumor): UACC62 (Melanoma); MCF7 (Normal mamma tumor); NCIADR (Mammatumor which expresses the phenotype resistance against multiple drugs)HT29 (Colon tumor); 786-O (Renal tumor); OVCAR-3 (Ovary tumor); PC-3(Prostate tumor); K-562 (Leukemia); ED₅₀ (effective doses 50 expressedin micrograms by milliliters μg/mL).

Remarks:

All these results can be considered excellent, if we take into referencethe ones published in the literature:

Banskota AH, et al. Chemical Constituents of Brazilian Propolis andtheir cytotoxic activities: J. Nat. Prod. 61, 896-900, 1998.

Banskota A H, et al. Two Novel Cytotoxic Benzofuran Derivatives fromBrazilian propolis; J. Nat Prod. 63, 1277-1279, 2000

Kimoto T, et al. Apoptosis and Suppression of tumor growth by ArtepillinC extracted from Brazilian Propolis; Cancer Detect, Prev. 22(6), 505-15,1998.

It is observed that most of our products presented a strongantiproliferate action in concentration ranges (in ppm) much lower thanthe ones shown by some of the isolated compounds of the BrazilianPropolis.

Cytotoxicity of some isolated compounds of Brazilian Propolis byBanskota and Kimoto are shown in FIG. 2.

We want to point out that the obtained products by our team consist ofraw material of our research project, from which we are obtaining newderivatives, guided by the principle of analogy and the results ofTOPS-MODE predictions.

Curves Concentration response of the tested compounds is shown in FIG.3.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a table of data of antitumoral activity shown by synthesizedcompounds.

FIG. 2 is a table of data concerning the cytotoxicity of some isolatedcompounds of Brazilian Propolis by Banskota and Kimoto.

FIG. 3 is a graph plotting percentage of growth versus concentration ofthe antitumoral activity of the synthesized compounds in FIG. 1.

FIG. 4 is a graph plotting percentage of growth versus concentration forthe antiproliferative activity of compound 37 or HB1.

FIG. 5 is a graph plotting percentage of growth versus concentration forthe response of Doxorubicin.

FIG. 6 is a graph of the antitumoral activity of1,5-bis(4-hydroxy-3-methoxyphenyl)-penta-1-4-dien-3-one.

FIG. 7 is a graph plotting percentage increase versus concentration forthe antitumoral activity of HB6.

FIG. 8 is a graph plotting percentage increase versus concentration forthe activity of HBMI.

FIG. 9 is a graph plotting percentage increase versus concentration forthe activity of HB5.

FIG. 10 is a table showing the results of tests of compound 37 on ananimal.

FIG. 11 is a graph detailing the evolution of an animal's body weightafter receiving administration of 2.5 g of compound 37 per Kg of bodyweight.

FIG. 12 is a table of data describing change in body weight from testsof 2.5 g of compound 37 per Kg of animal body weight.

FIG. 13 is a graph of the changes in animal body weight resulting fromadministration of 3.0 g of compound 37 per Kg of animal body weight.

FIG. 14 is a table showing changes in animal body weight resulting fromadministration of 4.0 g of compound 37 per Kg of animal body weight.

FIG. 15 is a graph of the changes in animal body weight resulting fromadministration of 4.0 g of compound 37 per Kg of animal body weight.

FIG. 16 is a table showing changes in animal body weight resulting fromadministration of 5.0 g of compound 37 per Kg of animal body weight.

FIG. 17 is a graph of the changes in animal body weight resulting fromadministration of 5.0 g of compound 37 per Kg of animal body weight.

FIG. 18 is a table of cell lines used in the assessment assays.

DETAILED DESCRIPTION OF THE INVENTION

37 compound, obtained by means of an organic synthesis procedure,presented cytostatic activity (growth inhibition) for all cell lines andcytotoxic activity (cellular death) for NCI460 (Lung), UACC62 (Melanoma)and MCF7 (Mamma) and NCIADR (Mamma resistant) from 0.25 μg/mL in thefirst antitumoral tests performed into CPQBA, IJNICAMP, Sep. 24, 2001.

Later, these tests of antiproliferate activity were extended to thefollowing cell liens:

Colon: Renal; Ovary; Prostate; Leukemia. A graph showing the results ofthese tests using compound 37 is shown in FIG. 4.

Curve Concentration response of 37.

The results of this compound were compared with Doxorubicin (commercialanticarcinogen used as a standard in these tests) being so similar andin some cases higher than this commercial anticarcinogen. A graphshowing these results is shown in FIG. 5.

Curve concentration response Doxorubicin.

For example, our product inhibited the growth of the Mama NCI-ADR cellline (the one that presents the phenotype of resistance against multipledrugs). This result becomes itself very interesting since Doxorubicin,utilized as a positive control, did not inhibit the growth of this humancell line.

The derivatives of1,5-bis(4-hydroxy-3-methoxyphenyl)-penta-1,4-dien-3-one shows theantitumoral activity identified in FIG. 6:

The sample denominated EHBI compound presented cytostatic activity forall cell lines from the concentration of 0.25 μg/mL and cytotoxicactivity from the concentration of 25 μg/mL, excepting NCI-ADR cell linewhich had only its growth inhibition around 25%. The results showed thissample was not selective for the studied cell lines.

The sample denominated HB6 compound presented cytostatic activity forall cell lines from the concentration of 25 μg/mL and cytotoxic activityin the concentration of 250 μg/mL, for HT-29, 786-0, NCI-ADR and K562cell lines. Furthermore, this sample presented cellular selectivity forHT-29 and 786-0 cell lines. Test data for HB6 is shown in FIG. 7.

The sample denominated HBMI compound presented cytostatic activity forall cell lines from the concentration of 0.25 μg/mL and only presentedcytotoxic activity for PC-03 and OVCAR-3 cell lines in the concentrationof 250 μg/mL. Test data for HBMI is shown in FIG. 8.

The sample denominated HB5 compound presented moderate cytostaticactivity for all cell lines from the concentration of 0.25 μg/mL and didnot present cytotoxic activity for any cell lines in the usedconcentrations. Test data for HB5 is shown in FIG. 9.

Also a toxicological test with the sample denominated 37 compound (AcuteToxicity I.D₅₀, via intraperitoneal). The LD₅₀ value, evaluated bylinear regression, was equal to 8.54 g/Kg, after 14 days of observation.That means this product can be considered itself, according to Loomis,in Principles of Toxicology, as practically nontoxic (compounds withLD₅₀ value between 5.0 and 15 g/Kg are considered practically nontoxic).

The table shown in FIG. 10 indicates an animal's body weight thatreceived 2.5 g/Kg of animal body weight of the product denominated “37COMPOUND,” administrated via intraperitoneal, in the beginning andending of the acute toxicity test.

The graph of FIG. 11 describes the evolution of the animal's body weightthat received 2.5 g/Kg of animal body weight of the product denominated“37 COMPOUND,” administrated via intraperitoneal, in the beginning andending of the acute toxicity test.

The table of FIG. 12 shows an animal's body weight that received 3.0g/Kg of animal body weight of the product denominated “37 COMPOUND,”administrated via intraperitoneal, in the beginning and ending of theacute toxicity test.

The graph of FIG. 13 describes the evolution of the animal's body weightthat received 3.0 g/Kg of animal body weight of the product denominated“37 COMPOUND,” administrated via intraperitoneal, in the beginning andending of the acute toxicity test.

The table of FIG. 14 shows an animal's body weight that received 4.0g/Kg of animal body weight of the product denominated “37 COMPOUND,”administrated via intraperitoneal, in the beginning and ending of theacute toxicity test.

The graph of FIG. 15 shows the evolution of the animal's body weightthat received 4.0 g/Kg of animal body weight of the product denominated“37 COMPOUND,” administrated via intraperitoneal, in the beginning andending of the acute toxicity test.

The table of FIG. 16 shows an animal's body weight that received 5.0g/Kg of animal body weight of the product denominated “37 COMPOUND,”administrated via intraperitoneal, in the beginning and ending of theacute toxicity test.

The graph of FIG. 17 shows the evolution of the animal's body weightthat received 5.0 g/Kg of animal body weight of the product denominated“37 COMPOUND,” administrated via intraperitoneal, in the beginning andending of the acute toxicity test.

Further this invention is illustrated by means of the following examplesof execution:

EXAMPLE 1 Preparation of 1,5-bis(4-hydroxy-3-methoxyphenyl)-penta1,4-dien-3-one. (Method 1)

From vanillin and acetone in a 2:1 molar rate in acid medium, intemperatures that changes between 25 and 60° C., under ultrasonicirradiation conditions in a range from 25 to 40 KHz for a period of 1 to3 hours further putting the reacted mixture into water/ice untilproducing the raw product, which dissolves in a solution of sodiumhydroxide or potassium hydroxide (between 10-30%) being filtered; thefiltrated is treated with hydrochloric acid or sulphuric acid from aconcentration between 10-30%, the obtained product being filtered again,finally it is washed with distillated water until obtaining a neutralpH, this operation being repeated until the total purification of theproduct, not being necessary to perform a new purification of thecompound using other procedures such as recrystallization orchromatographic column (the purity was determined by means of the HPLCtechnique):

Obtained 92% yield of the pure product. Melting point: 155-156° C.

Method of Preparation 2

A mixture formed of vanillin and acetone in a 2:1 molar rate in acidmedium laid during 5-8 days, in temperatures that changes between −10and 40° C., further putting the reacted mixture into water/ice untilproducing the raw product, which dissolves in a solution of sodiumhydroxide or potassium hydroxide (between 10-30%) being filtered: thefiltrated is treated with hydrochloric acid or sulphuric acid from aconcentration between 10-30%, the obtained product being filtered again,finally it is washed with distillated water until obtaining a neutralpH. This operation is repeated until the total purification of theproduct, not being necessary to perform a new purification of thecompound using other procedures such as recrystallization orchromatographic column (the purity was determined by means of the HPLCtechnique):

Obtained 89% yield of the pure product, melting point of 155-156° C.

EXAMPLE 2 Preparation of1,5-bis(3-metoxy-4-acetoxyphenyl)penta-1,4-dien-3-one1,5-bis(4-hydroxy-3-metoxyphenyl)-penta-1,4-dien-3-one.

It is stirred with an excess of acetic anhydride and sodium acetate.It's heated in a range of temperature between 20 and 110° C. for aperiod of time between 30 minutes to 3 hours. The obtained product isput into distillated water with ice. The precipitate product isrecrystallized with hot ethanol. Yield: 58%. Melting point: 150° C.

EXAMPLE 3 Preparation of1,5-Bis[3-metoxy-4-(3-methyl-but-2-eniloxy)phenyl]penta-1,4-dien-3-one.

A mixture formed of1,5-bis(4-hydroxy-3-metoxyphenyl)-penta-1,4-dien-3-one (2 mmol) in 10 mLof dimethylformamide and potassium carbonate (6 mmol) is stirred in arange of temperature between 20-50° C. during a period of time from 20to 60 minutes in inert atmosphere (argon or nitrogen). After that, 3mmol of 3-methyl-but-2-enyl bromide with constant stirring. Further, thestir is kept for another period of 5-8 hours with a stream of inert gaspouring all the mixture content into water with ice. It's extracted withchloroform in three occasions with approximately 3 mL of this solvent.The organic phase is washed with a solution of NaRSO₄ and then withdistillated water. The chloroform phase is dried with sodium sulfateanhydrous. After that, the solvent is filtered and rotoevaporated. Thepurification of the product is performed utilizing a chromatographiccolumn filled with silica gel and using a mixture formed oftoluene/ethyl acetate or n-Hexane/ethyl acetate as elution solvents inan appropriate rate. Yield: 53% of the oil liquid substance.

EXAMPLE 4 Preparation of1,5-Bis(3,4-dimetoxyphenyl)-penta-1,4-dien-3-one

Method 1

The mixture of 3,4-dimethoxy-benzaldehyd and acetone in equimolecularrate condition in presence of hydrochloric acid was submitted to aultrasonic bath in the frequence from 25 to 40 KHz between 10-60 minutesin a range of temperature between 25-60° C. Further the obtained productis put into distilled water and ice, filtering the precipitate andwashing it with distillated water. The aqueous phase is extracted withchloroform and washing the chloroform phase with distillated water andthen the chloroform phase is dried with sodium sulfate anhydrous,filtered and rotoevaporated. Yield: 87%.

Method 2

1,5-bis(4-hydroxy-3-metoxyphenyl)-penta-1,4-dien-3-one is stirred withan excess of dimethyl sulfate or methyl iodide in alkaline medium (KOHor NaOH), stirring it in a range of temperature between 25-50° C. duringa period of time that varies between 5-24 hours. The formed mixture isput into cold water, filtering the formed precipitate, neutralizing itwith HCl. Further, it is washed with water until neutral pH. The afterpurification of the product was not necessary. Yield: 85%.

EXAMPLE 5 Preparation of1,5-Bis(3,4-dimetoxyphenyl)-penta-1,4-dien-3-ylidenmalonitrile

In a mixture formed of 1,5-Bis(3,4-dimethoxyphenyl)-penta-1,4-dien-3-oneand malononitrile in equivmolecular rate condition, ammonium acetate,acetic acid and toluene are added following Cope's variant, heating inreflux for a period of time between 5-16 hours or followingKnoevenagel's third variant using piperidine as catalyzer. The obtainedproduct is put into distillated water and ice, filtering the precipitateand extracting the aqueous phase with chloroform and washing thechloroform phase with distillated water. Further, the chloroform phaseis dried with sodium sulfate anhydrous, filtered and rotoevaporated.Yield: 76%. Melting point: 216° C.

Methodology utilized to perform the antitumoral tests:

REPORT OF THE ANIPROLIFERATE TEST IN TUMORAL HUMAN CELLS WITH THESULFORRODAMINA B ESSAY

Cells

The cell lines used in the essays, described in Table 1, were maintainedin recipients of 25 cm² (Nunc®), with 5 mL of culture medium RPMI 1640supplemented with 5% bovine fetal serum (RPMI/SFB), at 37° C. inatmosphere of 5% of CO₂ and 100% of humidity.

TABLE 1 Cellular lineages used in the assessment essays of theantiproliferate activity Cellular Type Code Lung NCI460 Mama MCF-7 NCIADR* Melanoma UACC-62 Colon HT 29 Renal 786-0 Ovary OVCAR-3 ProstatePC-3 Leukemia K-562 *cell line that expresses the phenotype ofresistance against multiple drugs.

All of the procedures described below were performed under sterileconditions (Laminar Flux Veco®, Class IIB2).

Experimental procedure

100 mL of cells in medium RPMI/SFB with 50 μg/mL of Gentamicin wereinoculated in its respective densities of inoculation, in plates of 96compartments and incubated for 24 hours at 37° C. in atmosphere of 5% ofCO₂ and 100% of humidity.

After 24 hours, 100 mL of the test substance were added in the followingconcentrations of 250; 25; 2.5; 0.2 μg/mL diluted inRPMI/SFB/gentamicin. In this moment, the reading of one plate wasperformed for the determination of the T0 (cell control in the moment ofadding the samples). The other plates were incubated for 48 hours. Afterthis period, the experiment was paused by adding the trichlorine aceticacid to further determine the protein content by means of colorimetricessay with the sulforrodamina B.

Dilution of the samples

The stocked solutions were developed by diluting the samples in dimethylsulfoxide (DMSO) in the concentration of 0.1 g/mL. That solution wasdiluted by 400 times in RPMI/SFB/gentamicina to be added in the platesof 96 compartments being obtained to the ideal concentration of DMSO(Skehan e cols. 1990).

Essay of the Sulforrodamina B (SRB)

At the end of the test, the plates of 96 compartments were centrifugedfor 3 minutes at 2000 rpm, and they were fixed with 50 μL of a solutionat 50% of trichloroacetic acid (TCA) at 4° C. To complete the cellularfixation, the plates were incubated for 1 hour at 4° C.

The plates were submitted to four consecutive washes with distillatedwater to remove the residues of TCA, medium, SFB and secondary metabolicand maintained at room temperature until completely dry.

A coloration was performed by the adding of 50 μL SRB at 0,4%(weight/volume) diluted in acetic acid at 1%, and maintained at 4° C.for 30 minutes. Further, they were washed by 4 consecutive times with asolution of acetic acid 1%. The residue of the washing of the solutionwas removed and the plates were dried again at room temperature. Thecolorant linked to the cellular protein was diluted with a solution ofTrizma Base (Sigma®) in concentration of 10 ∞M and pH 10.5 for 5 minutesin ultrasonic bath. The spectrophotometric of absorbancy reading wasperformed in 560 nm in a micro plates lector (Labsystems Multiskan®MCC/340).

Analysis of the results

The absorbancy averages were assessed having discounted its respectivenulls and by means of the formula shown below was determined the growthinhibition (IC) of each tested sample.

T>C, the drug did not stimulate the growth, it does not present IG.

IF T≧T0 but <C, the drug was cyostatic and the formula used is100×[(T-To)/(C-T0)].

If T<T0, the drug was cytotoxic and the formula used is100×[(T-T0)/(C-T0)].

Considering that T is the absorbency average of the treated cell, C isthe cell control and T0 is the cell control in the addition day.

The obtained result was subtracted from 100% so obtaining the percentageof growth inhibition. The samples were considered actives since theypresented growth inhibition-dependent dose higher than 50% and selectivelineage, that is, the preferential activity for only one type of tumoralcell or with cytostatic and/or killed cell effect well distinguishedamong the cell lines.

All the assays were performed in triplicates so that the presentedresults make reference to a representative experiment. The standarddeflection from the average was always lower than 5%.

Methodology utilized to perform the toxicological tests:

Technique

* Ten albinic Swiss mice are used, of the male gender, weighingapproximate 25 g, for each of the treated groups and control.

* Adaptation period: the animals are maintained in the test room for atleast seven days before the beginning of the essay.

* The animals are submitted to a fast 12 hours before administering thetest substance, done by gavage, at the time the animal's body weight islisted.

* After the administration, the animals are maintained in observationfor a minimum period of 14 days.

* The number of dead animals for each one of the doses is listed and theLD₅₀ is assessed by Litchfield and Wilcoxon's (1949) method and theanimal's body weight is listed at the end of the acute toxicity test.

1. A process for preparing1,5-bis(4-hydroxy-3-methoxyphenyl)-penta-1,4-dien-3-one, characterizedin that it comprises contacting vanillin and acetone under ultrasonicirradiation.
 2. A process according to claim 1 characterized in thatvanillin and acetone are contacted in a mole ratio of 2:1.
 3. A processaccording to claim 1, characterized in that vanillin and acetone arecontacted at temperatures ranging from 25° C. to 60° C.
 4. A processaccording to claim 1, characterized in that the ultrasonic irradiationis in the range of from 25 to 40 KHz.
 5. A process according to claim 1,characterized in that vanillin and acetone remain in contact for aperiod of time ranging from 1 to 3 hours.
 6. A process according toclaim 1, characterized in that it additionally comprises purifying thepurification of 1,5-bis(4-hydroxy-3-methoxyphenyl)-penta-1,4-dien-3-oneobtained, mixing the reaction mixture in water/ice until a crude productis obtained, then dissolved in a sodium or potassium hydroxide solutionand filtered; the filtrate being treated with an acid selected from thegroup consisting of hydrochloric acid and sulfuric acid and additionalfiltration, successive washes with water being then carried out until aneutral pH is achieved.
 7. A process according to claim 6, characterizedin that the sodium or potassium hydroxide solution is at a concentrationbetween 10% and 30%.
 8. A process according to claim 6, characterized inthat the hydrochloric or sulfuric acid is at a concentration between 10%and 30%.
 9. A process for preparing1,5-bis(4-hydroxy-3-methoxyphenyl)-penta-1,4-dien-3-one, characterizedin that it comprises mixing vanillin and acetone in an acidic underultrasonic irradiation.
 10. A process according to claim 9,characterized in that it additionally comprises purifying1,5-bis(4-hydroxy-3-methoxyphenyl)-penta-1,4-dien-3-one obtained, mixingthe reaction mixture in water/ice until a crude extract is obtained,then dissolved in a sodium or potassium hydroxide solution and filtered;the filtrate being treated with hydrochloric or sulfuric acid andadditionally filtered, successive washes with water being carried outuntil a neutral pH is achieved.
 11. A process for preparing1,5-bis(3-methoxy-4-acethoxy-phenyl)-penta-1,4-dien-3-one, characterizedin that it comprises mixing1,5-bis(4-hydroxy-3-methoxyphenyl)-penta-1,4-dien-3-one, obtained by theprocess defined in claim 1, and acetic anhydride and sodium acetate. 12.A process of preparing1,5-bis(3-methoxy-4-acethoxyphenyl)-penta-1,4-dien-3-one, characterizedin that it comprises mixing1,5-bis(4-hydroxy-3-methoxyphenyl)-penta-1,4-dien-3-one, obtained by theprocess as defined in claim 1, in dimethylformamide and potassiumcarbonate, and then adding 3-methyl-but-2-enyl bromide.
 13. A processaccording to claim 10, characterized in that 3-methyl-but-2-enyl bromideis added to the mixture of1,5-bis(4-hydroxy-3-methoxyphenyl)-penta-1,4-dien-3-one.
 14. A processaccording to claim 13, characterized in that it additionally comprisespurifying1,5-bis[3-methoxy-4-(3-methyl-but-2-enyloxy)-phenyl]-penta-1,4-dien-3-one,putting said compound into water with ice, then extracting withchloroform, the washing the organic phase with NaHSO4 and then water;wherein the chloroform phase is dried with anhydrous sodium sulfate, andthen the solvent is filtered and rotoevaporated, and then the product ispassed through a chromatographic column filled with silica gel.
 15. Aprocess of preparing 1,5-bis(3,4-dimethoxy-phenyl)-penta-1,4-dien-3-one,characterized in that it comprises mixing 3,4-dimethoxybenzoaldehyde andacetone in an ultrasound bath.
 16. The process according to claim 15,characterized in that 3,4-dimethoxybenzoaldehyde and acetone are mixedin a ratio of 2:1.
 17. A process according to claim 15, characterized inthat it additionally comprises purifying the1,5-bis(3-,4-dimethoxy-phenyl)-penta-1,4-dien-3-one obtained, puttingwater with ice, filtering the precipitate, washing it with water,wherein the water phase is extracted with chloroform and the chloroformphase is dried with anhydrous sodium sulfate, filtered androtoevaporated.
 18. A process of preparing1,5-bis(3-,4-dimethoxy-phenyl)-penta-1,4-dien-3-one, characterized inthat is comprises mixing1,5-bis(4-hydroxy-3-methoxyphenyl)-penta-1,4-dien-3-one obtained, by theprocesses as defined in claim 1, wherein dimethyl sulfate or methyliodide.
 19. A process according to claim 18, characterized in that itadditionally comprises purifying1,5-bis(3,4-dimethoxy-phenyl)-penta-1,4-dien-3-one in ice-cold water,the formed precipitate is filtered, and then neutralized with HCl; thenthe product is washed with water until a neutral pH is achieved.
 20. Aprocess of preparing1,5-bis(4-hydroxy-3-methoxy-phenyl)-penta-1,4-dien-3 yliden-malonitryl,characterized in that it comprises mixing1,5-bis(4-hydroxy-3-methoxyphenyl)-penta-1,4-dien-3-one, obtained by theprocess as defined in claim 1 and malonitrile.
 21. Pharmaceuticalcomposition for the treatment of cancer, wherein said compositioncomprises at least one of the compounds obtained by the process definedin claim
 1. 22. A therapeutic method for the treatment of cancer,characterized in that one administers a therapeutically effective amountof a compound obtainable by the process as defined in claim 1 to asubject in need of such a treatment.